Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Abstract

Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest and Polyploidization

“…To this end, we used two single guide RNAs (herein dubbed gRNA), AXL-45 and AXL-185, targeting AXL gene locus at two different sites (exon 1 and exon 2; shown in Figure S1) and non-targeting Renilla gRNA (specific to Renilla luciferase gene) served as the negative control. Similar to the previously reported findings in SNU449 cell line [17],…”

“…To this end, we used two single guide RNAs (herein dubbed gRNA), AXL-45 and AXL-185, targeting AXL gene locus at two different sites (exon 1 and exon 2; shown in Figure S1) and non-targeting Renilla gRNA (specific to Renilla luciferase gene) served as the negative control. Similar to the previously reported findings in SNU449 cell line [17], AXL gene knock-out also proved relatively inefficient in all four HCC cell lines tested in this study. As compared to Renilla gRNA, AXL-185 gRNA caused a moderate decrease in AXL protein levels.…”

“…Indeed, to our knowledge, there is only one published example of studies related to phenotypic effects of AXL knock-out in the literature which has been reported in a mouse breast cancer cell line [16]. A previous attempt to inactivate the AXL gene by CRISPR/Cas9 technique in the SNU449 HCC cell line was unsuccessful [17]. Here we describe the successful inactivation of AXL expression in SNU475, a mesenchymal-like HCC cell line.…”

Gene Editing in Clinical Practice: Where are We?

“…This acts as a pair of 'molecular scissors' that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. a piece of RNA called guide RNA (gRNAThe protein Cas9 (or ''CRISPR-associated'') is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA [4,5]. CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence.…”

“…It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the medical field. The protein Cas9 (or ''CRISPR-associated'') is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA [4]. One would wonder how CRISPR works.…”